Magnus Åbrink

Rab27b regulates number and secretion of platelet dense granules

The Rab27 GTPase subfamily consists of two closely related homologs, Rab27a and Rab27b. Rab27a has been shown previously to regulate organelle movement and regulated exocytosis in a wide variety of secretory cells. However, the role of the more restrictedly expressed Rab27b remains unclear. Here we describe the creation of Rab27b knockout (KO) strain that was subsequently crossed with the naturally occurring Rab27a KO line, ashen, to produce double KO (Rab27aash/ash Rab27b−/−) mice. Rab27b KO (and double KO) exhibit significant hemorrhagic disease in contrast to ashen mice. In vitro assays demonstrated impaired aggregation with collagen and U46619 and reduced secretion of dense granules in both Rab27b and double KO strains. Additionally, we detected a 50% reduction in the number of dense granules per platelet and diminished platelet serotonin content, possibly due to a dense granule packaging defect into proplatelets during megakaryocyte maturation. The presence of Rab27a partially compensated for the secretory defect but not the reduced granule number. The morphology and function of platelet α-granules were unaffected. Our data suggest that Rab27b is a key regulator of dense granule secretion in platelets and thus a candidate gene for δ-storage pool deficiency in humans.

The Chymase, Mouse Mast Cell Protease 4, Constitutes the Major Chymotrypsin-like Activity in Peritoneum and Ear Tissue. A Role for Mouse Mast Cell Protease 4 in Thrombin Regulation and Fibronectin Turnover

To gain insight into the biological role of mast cell chymase we have generated a mouse strain with a targeted deletion in the gene for mast cell protease 4 (mMCP-4), the mouse chymase that has the closest relationship to the human chymase in terms of tissue localization and functional properties. The inactivation of mMCP-4 did not affect the storage of other mast cell proteases and did not affect the number of mast cells or the mast cell morphology. However, mMCP-4 inactivation resulted in complete loss of chymotryptic activity in the peritoneum and in ear tissue, indicating that mMCP-4 is the main source of stored chymotrypsin-like protease activity at these sites. The mMCP-4 null cells showed markedly impaired ability to perform inactivating cleavages of thrombin, indicating a role for mMCP-4 in regulating the extravascular coagulation system. Further, a role for mMCP-4 in connective tissue remodeling was suggested by the inability of mMCP-4 null peritoneal cells to process endogenous fibronectin.

Critical Role of Mast Cell Chymase in Mouse Abdominal Aortic Aneurysm Formation

Background— Mast cell chymase may participate in the pathogenesis of human abdominal aortic aneurysm (AAA), yet a direct contribution of this serine protease to AAA formation remains unknown. Methods and Results— Human AAA lesions had high numbers of chymase-immunoreactive mast cells. Serum chymase level correlated with AAA growth rate (P=0.009) in a prospective clinical study. In experimental AAA produced by aortic elastase perfusion in wild-type (WT) mice or those deficient in the chymase ortholog mouse mast cell protease-4 (mMCP-4) or deficient in mMCP-5 (Mcpt4−/−, Mcpt5−/−), Mcpt4−/− but not Mcpt5−/− had reduced AAA formation 14 days after elastase perfusion. Even 8 weeks after perfusion, aortic expansion in Mcpt4−/− mice fell by 50% compared with that of the WT mice (P=0.0003). AAA lesions in Mcpt4−/− mice had fewer inflammatory cells and less apoptosis, angiogenesis, and elastin fragmentation than those of WT mice. Although KitW-sh/W-sh mice had protection from AAA formation, reconstitution with mast cells from WT mice, but not those from Mcpt4−/− mice, partially restored the AAA phenotype. Mechanistic studies suggested that mMCP-4 regulates expression and activation of cysteine protease cathepsins, elastin degradation, angiogenesis, and vascular cell apoptosis. Conclusions— High chymase-positive mast cell content in human AAA lesions, greatly reduced AAA formation in Mcpt4−/− mice, and significant correlation of serum chymase levels with human AAA expansion rate suggests participation of mast cell chymase in the progression of human and mouse AAA.

Mast cells regulate homeostatic intestinal epithelial migration and barrier function by a chymase/Mcpt4-dependent mechanism.

Altered intestinal barrier function is postulated to be a central predisposing factor to intestinal diseases, including inflammatory bowel diseases and food allergies. However, the mechanisms involved in maintaining homeostatic intestinal barrier integrity remain undefined. In this study, we demonstrate that mice deficient in mast cells (KitW-sh/W-sh [Wsh]) or mast cell chymase (Mcpt4−/−) have significantly decreased basal small intestinal permeability compared with wild-type (WT) mice. Altered intestinal barrier function was linked to decreased intestinal epithelial cell migration along the villus/crypt axis, altered intestinal morphology, and dysregulated claudin-3 crypt expression. Remarkably, engraftment of Wsh mice with WT but not Mcpt4−/− mast cells restored intestinal epithelial cell migration, morphology, and intestinal epithelial barrier function. Collectively, these findings identify a mechanism by which mast cells regulate homeostatic intestinal epithelial migration and barrier function.

Rab27b Regulates Mast Cell Granule Dynamics and Secretion

The Rab GTPase family regulates membrane domain organization and vesicular transport pathways. Recent studies indicate that one member of the family, Rab27a, regulates transport of lysosome‐related organelles in specialized cells, such as melanosomes and lytic granules. Very little is known about the related isoform, Rab27b. Here we used genetically modified mice to study the involvement of the Rab27 proteins in mast cells, which play key roles in allergic responses. Both Rab27a and Rab27b isoforms are expressed in bone marrow‐derived mast cells (BMMC) and localize to secretory granules. Nevertheless, secretory defects as measured by β‐hexosaminidase release in vitroand passive cutaneous anaphylaxis in vivowere found only in Rab27b and double Rab27 knockout (KO) mice. Immunofluorescence studies suggest that a subset of Rab27b and double Rab27‐deficient BMMCs exhibit mild clustering of granules. Quantitative analysis of live‐cell time‐lapse imaging revealed that BMMCs derived from double Rab27 KO mice showed almost 10‐fold increase in granules exhibiting fast movement (>1.5 μm/s), which could be disrupted by nocodazole. These results suggest that Rab27 proteins, particularly Rab27b, play a crucial role in mast cell degranulation and that their action regulates the transition from microtubule to actin‐based motility.

Neurotensin increases mortality and mast cells reduce neurotensin levels in a mouse model of sepsis.

Sepsis is a complex, incompletely understood and often fatal disorder, typically accompanied by hypotension, that is considered to represent a dysregulated host response to infection. Neurotensin (NT) is a 13-amino-acid peptide that, among its multiple effects, induces hypotension. We find that intraperitoneal and plasma concentrations of NT are increased in mice after severe cecal ligation and puncture (CLP), a model of sepsis, and that mice treated with a pharmacological antagonist of NT, or NT-deficient mice, show reduced mortality during severe CLP. In mice, mast cells can degrade NT and reduce NT-induced hypotension and CLP-associated mortality, and optimal expression of these effects requires mast cell expression of neurotensin receptor 1 and neurolysin. These findings show that NT contributes to sepsis-related mortality in mice during severe CLP and that mast cells can lower NT concentrations, and suggest that mast cell–dependent reduction in NT levels contributes to the ability of mast cells to enhance survival after CLP.

Mast cell chymase reduces the toxicity of Gila monster venom, scorpion venom, and vasoactive intestinal polypeptide in mice

Mast cell degranulation is important in the pathogenesis of anaphylaxis and allergic disorders. Many animal venoms contain components that can induce mast cell degranulation, and this has been thought to contribute to the pathology and mortality caused by envenomation. However, we recently reported evidence that mast cells can enhance the resistance of mice to the venoms of certain snakes and that mouse mast cell-derived carboxypeptidase A3 (CPA3) can contribute to this effect. Here, we investigated whether mast cells can enhance resistance to the venom of the Gila monster, a toxic component of that venom (helodermin), and the structurally similar mammalian peptide, vasoactive intestinal polypeptide (VIP). Using 2 types of mast cell-deficient mice, as well as mice selectively lacking CPA3 activity or the chymase mouse mast cell protease-4 (MCPT4), we found that mast cells and MCPT4, which can degrade helodermin, can enhance host resistance to the toxicity of Gila monster venom. Mast cells and MCPT4 also can limit the toxicity associated with high concentrations of VIP and can reduce the morbidity and mortality induced by venoms from 2 species of scorpions. Our findings support the notion that mast cells can enhance innate defense by degradation of diverse animal toxins and that release of MCPT4, in addition to CPA3, can contribute to this mast cell function.

Serotonin and histamine storage in mast cell secretory granules is dependent on serglycin proteoglycan

BACKGROUND Serotonin and histamine are components of human and rodent mast cell secretory granules. OBJECTIVE Serotonin and histamine are stored in the same compartment as serglycin proteoglycan. Here we addressed the possibility that serglycin may be involved in their storage and/or release. METHODS The storage and release of histamine and serotonin was studied in bone marrow-derived mast cells (BMMCs) and in peritoneal mast cells from wild-type or serglycin-/- mice. RESULTS Both serotonin and histamine storage in BMMCs was positively correlated with the degree of mast cell differentiation, and the amount of stored amine was reduced in serglycin-/- BMMCs compared with wild-type controls. The amounts of histamine/serotonin stored were reflected by the expression levels of histidine decarboxylase and tryptophan hydroxylase 1, respectively. Calcium ionophore activation resulted in serotonin/histamine release both from wild-type and serglycin-/- BMMCs. Interestingly, serotonin release was induced in cells lacking intracellular stores of serotonin, suggesting de novo synthesis. The knockout of serglycin affected the levels of stored and released mast cell serotonin and histamine to an even larger extent in in vivo-derived mast cells than in BMMCs. CONCLUSION These results establish a previously assumed, but not proven, role of serglycin in storage of histamine and, further, establish for the first time that serotonin storage in mast cells is dependent on serglycin proteoglycan.

Serglycin proteoglycan is required for secretory granule integrity in mucosal mast cells

SG (serglycin) PGs (proteoglycans) are strongly implicated in the assembly of MC (mast cell) granules. However, this notion has mainly been on the basis of studies of MCs of the connective tissue subtype, whereas the role of SG PG in mucosal MCs has not been explored. In the present study, we have addressed the latter issue by using mice with an inactivated SG gene. Bone marrow cells were differentiated in vitro into the mucosal MC phenotype, expressing the markers mMCP (mouse MC protease) -1 and -2. Biosynthetic labelling experiments performed on these cells revealed an approximately 80% reduction of 35SO4(2-) incorporation into PGs recovered from SG-/- cells as compared with SG+/+ counterparts, indicating that SG is the dominating cell-associated PG of mucosal MCs. Moreover, the absence of SG led to defective metachromatic staining of mucosal MCs, both in vivo and in the in vitro-derived mucosal MCs. Ultrastructural analysis showed that granules were present in similar numbers in SG+/+ and SG-/- cells, but that their morphology was markedly affected by the absence of SG, e.g. with electron-dense core formation only seen in SG+/+ granules. Analysis of the MC-specific proteases showed that mMCP-1 and mMCP-7 were completely independent of SG for storage, whereas mMCP-2 showed a partial dependence. In contrast, mMCP-4 and -6, and carboxypeptidase A were strongly dependent on SG for storage. Together, our data indicate that SG PG is of crucial importance for assembly of mature mucosal MC granules, but that the specific dependence on SG for storage varies between individual granule constituents.

Independent degeneration of photoreceptors and retinal pigment epithelium in conditional knockout mouse models of choroideremia

Choroideremia (CHM) is an X-linked degeneration of the retinal pigment epithelium (RPE), photoreceptors, and choroid, caused by loss of function of the CHM/REP1 gene. REP1 is involved in lipid modification (prenylation) of Rab GTPases, key regulators of intracellular vesicular transport and organelle dynamics. To study the pathogenesis of CHM and to develop a model for assessing gene therapy, we have created a conditional mouse knockout of the Chm gene. Heterozygous-null females exhibit characteristic hallmarks of CHM: progressive degeneration of the photoreceptors, patchy depigmentation of the RPE, and Rab prenylation defects. Using tamoxifen-inducible and tissue-specific Cre expression in combination with floxed Chm alleles, we show that CHM pathogenesis involves independently triggered degeneration of photoreceptors and the RPE, associated with different subsets of defective Rabs.